1-benzyl tetramic acid derivatives



United States Patent This invention relates to novel amino acidderivatives and more particularly to novel tetramic acids and saltsthereof. substituted in the 1-, 3-, and S-positions and salts thereof.

The compounds of the present invention have the following structuralformula:

where R is alkyl, haloalkyl, or aryl, R is benzyl, phenyl, or alkyl, R"is acetyl, carboxamido, or carboethoxy, X is hydrogen or a metal cation,and n is a whole number corresponding to the valency of X. 7

Representative of the compounds within the generic formula appearingabove are l-methyl-3-acetyl-S-sec. butyl tetramic acid,l-benzyl3-acetyl-5-isopropyl tetramic acid, l,5-diphenyl-3acetyltetramic acid, l-benzyl-3-acetyl-5-(3'-amyl) tetramic acid,l-benzyl-S-acetyl-S-sec. butyl tetramic acid, l-benzyl-3-acetyl-5- 3 3 3-trifluoro-n-propyl) tetramic acid, l-benzy1-3-acetyl-5-(n-amyl)tetramic acid, 1-benzyl-3-acetyl-5-'phenyl tetramic acid,1-benzyl-3-carboxamido-5-sec. butyl tetramic acid,1-benzyl-3-carboethoxy-5-sec. butyl tetramic acid, and the sodium,potassium, calcium, magnesium, copper, and barium salts of theforegoing, such as, for example, potassium-1-methyl-3-acetyl-5-sec.buty-l tetramate, sodium- 1 -benzyl-3-acetyl-5 -isopropy1 tetramate,sodiuml-benzyl-3-acetyl-5-sec. butyl tetramate,

potassium-l-methyl-3-acetyl-5-sec. butyl tetramate,

copper-l-benzyl-3-carboethoxy-5-sec. butyl tetra-mate,magnesium-1,5diphenyl-3-acetyl tetramate, and the like. The compoundsmay be either in the L form, D form, or mixtures of these.

The preferred compounds of the present invention are those wherein R isbenzyl, R is acetyl, and R is lower alkyl or halo lower alkyl, such asthe following:

l-benzyl--3-acetyl-5-sec. butyl tetramic acid,

l-benzyl-3-acetyl5-n-hexyl tetramic acid,

1-benzyl-3-acetyl-5-(3-amy1) tetramic acid,

1-benzyl-3-acetyl-5-isopropyl tetramic acid,

l-benzyl-3-acetyl-5-(333'-trifluoro-n-propyl) tetramic acid,

1-benzyl-3-acetyl-5-(n-amyl) tetramic acid, and the sodium and potassiumsalts thereof.

The compounds of the present invention are prepared from theappropriately substituted amino acid in accordance with the flow diagramappearing hereinafter. The starting material is an amino acid of thefollowing formula Specifically it relates to novel tetramic acids I Asthe foregoing diagram indicates, the series of steps introduces into thestarting amino acid the R and R groups. g

With respect to the amino acid starting material, where R is sec. butyl,isoleucine 'is the" starting material; where R is isopropyl, valine isused;.where R is 3-amyl, 3,3-diethyl alanine is the starting material;where R is 3'33'-trifiuoro-n-propyl, the starting rnaterial is5,5,5-trifluoro norvaline and so forth. In some instances the selectionof the starting material may also be made on the basis of it alreadycontaining an appropriate R group, thereby eliminating the first stepshown in the flow sheet. For example, the starting material forl-benzyl-3-acetyl-5-isopropyl tetramic acid is conveniently N-benzylvaline; for 1-methyl-3-acetyl-5-sec. butyl tetramic acid, the startingamino acid is N-methyl isoleucine. Thus, it can be seen that wherepossible and where practicable the starting material may be one whichincludes both R and R initially. Furthermore, the starting materialsneed not be in any particular form with respect to optical activity.Thus, either D or L forms, or mixtures of these may 'be 'employed. I

For starting materials having no N-substituent, the first step of theprocess involves introducing at the nitro-' gen the R substitutent. Inthe case of introducing the benzyl radical, the amino acid, preferablyin the form of the sodium or potassium salt, is reacted withbenzaldehyde in a solvent, and the resulting mixture'hydrogenated' atroom temperature using, for example, paladium on carbon as a catalyst.Low hydrogen pressure, of the order of 040 p.s.i.g., is suitable. When Ris an alkyl radical from ethyl and higher, such radical may beintroduced by employing the appropriate aldehyde. When R is methyl orphenyl, the appropriate amino acid starting ma terial containing thegroups is employed and may be pre-i tion toconvert to the free acid, theacetoacetyl derivative may be'recovered'from the reaction mass byextracting it into ether. When R" is carboethoxy, carbethoxy acetylchloride is used in place of diketene.

The next step involves esterifiction of the N-acetoacetyl free acidusing diazomethane, preferably in excess, and in the presence of asuitable solvent such as ether, diethyl ether, and the like, andpreferably at 20 C. The product obtained thereby is the methyl ester ofthe N-disubstituted amino acid starting material and is obtained as anoily residue from the reaction mixture after evaporation of the solvent.

The methyl ester obtained above is then treated to effect ring closureto form Compounds I. This is achieved by treating the ester with analkaline condensing agent such as sodium, potassium, magnesiumalkoxides, and the like with, sodium and potassium methoxide beingpreferred, at a temperature preferably in the range of from 70 C. to 100C. in the presence of a suitable solvent such as benzene, toluene, andthe like. The product, Compounds I, is obtained as the metal saltcorresponding to the metal present in the condensing agent. ,They may beconverted to thefree acid by acidifying the salt in an aqueous solutionThe soluble metal salt's'may'be converted to insoluble metal salts suchas the copper salt, by the addition of appropriate metal salt solutions,or converted to free acid form by acidification. The free acidsfrequently can be crystallized from solvents such as ether,

ethyl acetate, benzene petroleum ether, and mixtures aureus, Bacillusszlbiilis,St aphlococcu s aw cus, Bacillus megaterium, Streptococcusfaecalis, and Salmonella gallinarum. l p

' The following examples are given for the purpose of illustration onlyand not by way of limitation.

EXAMPLE 1 I-bcnzyl-3-acelyl-5-(3'-anzyl) tetmmic acid 5.5 g. ofdiethylalanine are mixed with 3.6 g. of benz-' aldehyde in methanol withone equivalent of potassium hydroxide and this mixture is hydrogenatedin the'presence of a palladium catalyst at room temperature, start: ingat 40 p.s.i.g. The reaction is complete when one molar equivalent ofhydrogen-based on-benzaldehyde is absorbed. The catalyst is then removedby filtration, and the solution is neutralized with concentratedhydrochloric acid, and filtered quickly to remove the precipitatedpotassium chloride. N-benzyl diethylalanine crystallizes from thefiltrate in 6.86 g.- yield. Melting point 240242 C. with sublimation.

A Izalysis.-C H NO calc.: C, 71.45; H, 8.99; N, 5.95. Found: C, 71.76;H, 11.44; N, 5.32.

2.35 g. of N-benzyl diethylalanine is dissolved in one equivalent ofsodium hydroxide and cooled in an ice bath. 1.82 g. of diketeneis addedwith agitation. After one hour the solution is extracted with ether.Crystalline N-acetoacetyl-N-benzyl diethylalanine is recovered byacidifying the aqueous solution with dilute hydrochloric acid. Afterrecrystallization from ethyl acetate, the product has a melting point of129-131 C.

Analysis.C H NO calc.: C, 67.69; H, 7.89; N, 4.39. Found: C, 67.78; H,7.96; N, 4.11.

A quantity of the above acid, 3.06 g., is converted to the methyl esterby treating with an excess of diazomethane in ether solution, whilecooled in an ice water bath,

' times.

until a yellow color remains. The excess diazomethane is evaporatedunder a stream of nitrogen and the ether concentrated to yield the oilyester. Approximately 0.0115 moles of sodium in 5 ml. methanol is addedto the acetoacetyl ester in 20 ml. benzene and the solution refluxed ona steam bath for 3 hours. The benzene is decanted and the residuetriturated with ether to give a crude sodium salt. The crude sodium saltof the product, 1- benzyl-3-acetyl-5-(3-amyl) tetramic acid, isdissolved in water, neutralized with sulfuric acid to obtain the freeacid which is then extracted into ether. The ether extract is washed,dried, and reconverted to the sodium salt by back extracting with acalculated amount of sodium bicarbonate. The aqueous layer islyophilized to yield the solid sodium salt. U.V. in methanol at pH 7Amax. 2480 and 2850. At pH 1 xmax. 2830.

EXAMPLE 2 Potassium-1 -metl1yl-3-acety I-S-sec. bulyl tetra/1 rate 3.5g. (.024 mole) of N-methyl L-isoleucine in 9.7 ml. of 2.5 N NaOH inabout 50 ml. of water is cooled in an ice bath and 2.23 g. of diketeneadded thereto. At this time the solution is neutral. The solution isextracted once with ether and the aqueous solution acidified andextracted three times with ether. The ether extracts from the acidifiedaqueous solution are combined and washed twice with water, dried withmagnesium sulfate, and concentrated. Yield is 5 g. of N-acetoacetyl N-methyl L-isoleucine.

- Analysis.C H N O calc.: C, 65.30; H, 8.36; N, 8.02. Found: C, 65.52;H, 8.14; N, 7.95.

The above product is next converted into the methyl ester by reactionwith an excess of diazomethane in an ice bath. The resulting solution isdried over magnesium sulfate and concentrated to yield 5 g. of themethyl ester of the foregoing acid. This is then dissolved in ,15 ml. ofmethanol and 15 ml. of benzene. A solution of 0.54 g.

- of sodium in about 5 ml. of methanol is added and the mixture refluxedfor three hours. The solution is concentrated and the productprecipitated with ether. The sticky residue is dissolved in water,acidified with hydrochloric acid, and extracted with petroleum etherthree The ether solution is washed with water until free of hydrochloricacid, dried over magnesium sulfate, and potassium-Z-ethylhexanoate inmethyl isobutyl kc tone added thereto. A precipitate thereupon formswhich is triturated with ether and then with acetone to yield 2.87 g. ofcrystalline potassium-l-methyl-3-acetyl-5-sec. butyl tetramate.

Analysis.C H NO K, calc.: C, 53.03; H, 6.46; N, 5.62. Found: C, 53.21;H, 6.35; N, 5.43.

EXAMPLE 3 I-benzyl-3-ace1yl-5-is0propyl lelramic acid 5.85 g. (0.05mole) of valine is dissolved in about ml. of methanol containing 2.8 g.(0.05 mole) of potassium hydroxide. 5.3 g. (0.05 mole) benzaldehyde isadded and the solution is shaken with hydrogen at room temperature in apressure bottle using palladium on carbon as a catalyst, following theprocedure of Example 1. The product starts to crystallize in about 3hours and the hydrogenation is terminated. The precipitate isredissolved by heating the reaction mixture on a steam bath, and theclear solution filtered from the catalyst and neutralized with oneequivalent of concentrated hydrochloric acid to give immediatecrystallization of N-benzyl L- 1 valine in 91% yield.

' tion is extracted once with ether and the remaining aqueous solutionacidified with dilute hydrochloric acid. The

EXAMPLE 4 N-benzyl-3-acelyl-5=sec. butyl tetramic acid The N-benzylderivative of L-isoleucine is prepared by arat-es as an oil and isextracted int-o ether. The ether extract is washed with dilutehydrochloric acid and water, dried over magnesium sulfate, andconcentrated to yield 17 g. (47% overall yield) of the diethyl ester ofcarboxyacetyl isoleucine.

17 g. of the above ester, is treated with 1.26 g. of sodium dissolved inml. of ethanol in 50 ml. of benzene.

After refluxing for three hours, the solution is decanted, the residuedissolved in water, acidified with hydrochloric following the proceduregiven in Example 3, using equivalent amounts of L-isoleucine in place ofvaline. Acetoacetylation, esterification', and ring closure are effectedas therein indicated. The product, N-benzyl-3-acetyl-5- sec. butyltetramic acid, potassium salt, has the following characteristics: U.V.in MeOH Amax. at pH 7, 2470, 2840; Mnax. at pH 1, 2820.

EXAMPLE 5 1-benzyl-3-acefyI-5-(3'3'3-trifluor0-n-pr0pyl) tetramic acid1-benzyl-3-acetyl-5-(333-trifiuoro-n-propyl) tetramic acid is preparedin the same manner as described in Example 3, using equivalent amountsof 5,5,5,-trifiuoro norvaline instead of valine. The product has amelting point of from 73-76 C.

EXAMPLE 6 1-benzyl-3-acetyl-5-pl1enyl tetramic acid 5.49 g. of a-phenylglycine is dissolved in 150 ml. of methanol in which 2.2 g. of potassuml'lYl'OXidC, is present. 4.2 g. of benzaldehyde is added and the mixtureis hydrogenated with platinum catalyst in accordance with the procedureof Example 1. The resulting solution is filtered from the catalyst andneutralized with concentrated hydrochloric acid to yieldN-benzyl-a-phenyl glycine as a crystalline precipitate. Afterrecrystallization with methanol, the precipitated N-benzyl-a-phenylglycine has a melting point of about 225 C.

2.27 g. of the above product is dissolved in 50 ml. of water containing0.8 g. of sodium hydroxide. 0.84 g. of diketene is added to the solutionwhile stirring in an ice bath. After /2 hour, the solution is extractedwith ether and the aqueous solution is acidified to yieldN-acetoacetyl-N-benzyl-a-phenyl glycine as a precipitate.

Analysis.C H NO calc.: C, 65.95; H, 7.27; N, 4.81. Found: C, 65.67; H,7.01; N, 5.05.

The above product is esterified in ether with diazomethane and ringclosure efl'ected as described'in Example 2. The product,l-benzyl-3-acetyl-5 phenyl tetramic acid, has the followingcharacteristics. Analysis: C H NO calc.: C, 74.25; H, 5.58; N, 4.56.Found: C, 74.40; H, 5.67; N, 4.18 U.V. in MeOH Amax. at pH 7, 2490,2860; Xmax. at pH 1, 2830.

EXAMPLE 7 I-benzyI-3-carb0eth0xy-5-sec. butyl tell-antic acidN-benzyl-isoleucine is esterified by dissolving the acid in ethanol towhich 100% excess of thionyl chloride has been added based on the amountof acid and refluxing for two hours. The resulting solution isconcentrated by vacuum, the residual ethyl ester hydrochloride isdissolved in ether and is crystallized by adding benzene thereto; M.P.,185 C. with sublimation.

To 29 g. of Nabenzyl-isoleucine ethyl ester hydrochloride in 100 ml. ofpyridine, cooled in an ice :bath, 15.4 g. of carboethoxy acetyl chlorideis added dropwise. After stirring for about Aa-hour, the mixture isconcentrated and the residue taken up in ice water. The product sepacidand extracted into ether. The ether solution is washed with water, driedover magnesium sulfate, and concentrated to yield 12.3 g. (86% yield) of1-benzyl-3- carboethox-y-S-sec. butyl tetramic acid; M.P., 185l92 C.with sublimation. The copper salt of the above acid is prepared bydissolving the acid in one equivalent of sodium hydroxide and treatingthe solution dropwise with aqueous cupric acetate. A green precipitateforms which is recrystallized from ethanol-Water mixture.

Analysis.C H NO /2Cu, calc.: C, 60.53; H, 6.21; N, 3.92. Found: C,60.48; H, 6.17; N, 3.67.

EXAMPLE 8 J-benzyI-3-carbOxz1mid0-5-sec. butyl letramic acid 1 g. of thecarboethoxy compound derivative produced in Example 7 is heated for 12hours in 40 ml. of liquid ammonia and 7.5 ml. of ethanol at C. in apressure bomb. The resulting solution is concentrated to dryness and theresidue dissolved in water, acidified with hydrochloric acid, andextracted into ethylaoetate. The resultingsolution is dried withmagnesium sulfate and concentrated to dryness. The residue,l-benzyl-3-carboxamido-S-sec. butyl tetramic acid is recrystallized fromether and has a melting point of 148 C.

Analysis.C I-I N O calc.: C, 66.64; H, 6.95; N, 9.72. Found: C, 66.22;H, 6.73; N, 9.68.

EXAMPLE 9 1,5-diphenyl-3-acetyl telramic acid 4.67 g. ofethyl-a-anilino-a-phenylacetate is dissolved in benzene, treated with1.74 g. of diketene, heated on a steam bath and allowed to standovernight. The solution is then "concentrated to an oily residue whichis washed with hot hexane to yield about 250 mg. of N-aceto-acetyl-N-phenyl-a-phenyl glycine, ethyl ester. 700 mg. of this ester is ringclosed by refluxing the ester with one equivalent of magnesium dissolvedin 0.5 ml. of ethanol all in 10 ml. of benzene. After concentration todryness and acidification of the residue with dilute hydrochloric acid,the product, 1,5-diphenyl-3-acetyl tetramic acid, is obtained; M.P.,136138 C.

Analysis.--C H NO calc.: C, 73.70; H, 5.16; N, 4.77. Found: C, 74.19; H,5.40; N, 4.83. M.W.: 293.33.

EXAMPLE 10 The antibacterial activity of the various compounds listedbelow is determined by using the assay method of E. O. Stapley, AppliedMicrobiology, 6, 392-8 (1958). The compounds are assayed as aqueoussolutions at a concentration of 2 mg./ml. in pH 5, M/20 phosphatebuffer. Five m1. of nutrient agar containing 0.2% of yeast extract isplaced in a Petri dish and seeded with the micro organism indicated inTable I below. A fii-inch (6.4 mm.) paper disc is placed on the agar,soaked with droplets of the aqueous solutions discussed above, and theentire assembly allowed to incubate overnight at 25 C. The area of theagar medium in which microorganism growth occurs appears cloudy, whereasthe area in which no growth occurs remains clear. The diameter of theclear zones are indicative of relative antibacterial efficacy for thecompounds tested. The numerical designations in Table I below arediameters of zones of inhibitions.

TABLE L-ANTIBACTERIAL ACTIVITY Zones oi Inhibition (Diameters in mm.,H=Hazy) I II III IV V VI VII VIII IX X Escherichia coli 07 07 00 00 0000 00 00 12 Bacillus sp 23 26 28 27 26 31 28 20 37 28 Proteus vulgari 0007 07 00 B16 00 00 ()0 00 11 Pseudomonas aeruginusa 00 07 07 00 00 00 0000 00 00 Scrrntiu marcescc'ns 00 07 07 00 00 00 00 O0 ()0 10 Staph.aureus H13 25 28 28 24 34 34 17 33 29. Bacillus suhnzis 16 2s 2s 29 2835 29 1a 33 28 Sarcina lutea 11 19 24 23 29 29 29 28 29 Staph. mucus(Strep. resistant strain) 0O 22 23 28 22 32 27 13 3O 24 Strep. faecalis13 15 17 20 22 22 22 00 23 19 00 11 0s 1s 20 11 11 14 is 12 00 07 07 9011 10 10 00 00 11 Vibriu perc0lans 11 10 10' 15 1a 21 12 13 15 10Xanthomonas vcsicatoria 00 07 07 00 B10 00 I111 00 ()0 H11 Compoundsused in this example: What is claimed is:

20 1. A compound having the following structural for-IPotass1um-l-methyl-3-acetyl-5-sec. butyl tetramate mulaII-Potassium-1-benzyl-3-acetyl-5-isopropyl tetramateIII-Potassium-l-benzyl-3-acetyl-5- (3 -amyl) tetrarnateIVPotassium-l-benzyl-3-acetyl-5-sec. butyl tetramate 1 2:Vl-Benzyl-3-acetyl-5-(3'3'3-trifluoro-n-propyl) tetra- 25 N mic acid X+(3H2 VISodium-l-benzyl-3-acetyl-5-arnyl tetramateVIIPotassium-l-benzyl-3-acetyl-5-phenyl tetramateVIII-Copper-l-benzyl-B-carboethoxy-S-sec. butyl tetramate V nIXPotassium-l-benzyl-3-acetyl-5-n-hexyltetramateXSodium-1-benzyl-3-carboxamido-5-sec. butyl tetramate As demonstrated inthe table, the compounds of the invention have a broad spectrum ofantibacterial activity. They also present a good variety of eificacy onparticular species. For example, while Compound III shows relativelylittle effect on Brucclla branchiseptica, Compounds IV and V show verygood activity against this microorganism.

EXAMPLE 11 TABLE II Wt. of Com pound Added to Disc, Gannnas Zonediameter, 111111.

Compound Micro organism B. meyalcrz'um 1, 200 600 B.s1lblilis B.sul/lilis .c

Any departure from the above description which conforms to the presentinvention is intended to be included within the scope of the claims.

wherein R is selected from the group consisting of loweralkyl,haloloweralkyl and phenyl; R" is selected from the group consisting ofacetyl, carboxamido, and carboethoxy; X is selected from the groupconsisting of hydrogen and Periodic Chart Group I and Group IIA metalcation; and n is a whole number corresponding to the valency of X.

2. A compound having the following structural formula wherein R isloweralkyl; R" is selected from the group consisting of acetyl,carboxamido, and carboethoxy; X is selected from the group consisting ofhydrogen and Periodic Chart Group I and Group IIA metal cation; and n isa whole number corresponding to the valency of X.

3. A compound having the following structural formula wherein R isloweralkyl; R is selected from the group consisting of acetyl,carboxamido, and carboethoxy; X

is an alkali metal; and n is a whole number corresponding to the valencyof X.

9 10 4. A compound having the following structural for- 10. 1benzyl-3-acetyl-5-(3'3'3'-trifluoro'n-propyl) temula tramic acid.

0 11. 1-benZyl-3-carbethoxy-5-sec. butyl tetrarnic acid. II 12.l-benzyl-3-carb0xamido-5-sec. butyl tetramic acid. 0 CH: 5

5 1 References Cited by the Examiner X+ 1 UNITED STATES PATENTS OH:2,660,583 11/1953 Lacey 260-3265 2,953,491 9/1960 Hardy et al. 16733 103,105,080 9/1963 Hickner 260326.3 3,153,656 10/1964 Biel 260-326.3 113,169,904 2/1965 Calderbank 167-33 wherein R is loweralkyl; X is analkali metal; and n is a THER REFERENCES Whole number Corresponding tothe valency Of Isowa et al., Bull. Chem. Soc. Japan, vol. 35, pp. 194145. 1-benzyl-3-acetyl-5-a1kyl tetramic acid. (1962). 6. An alkali metalsalt of 1-benzyl-3-acetyl-5-alkyl tetramic acid. ALEX MAZEL, PrimaryExaminer.

7. l-benzyl-3-acetyl-5-sec. butyl tetramic acid. 8.Sodium-l-benzyl-3-acetyl-5-sec. butyl tetramate. 20 HENRY JILES Examiner9. Potassium-1-benzyl-3-acetyl-5-sec. butyl tetramate. J A. NARCAVAGE,Assistant Examiner.

1. A COMPOUND HAVING THE FOLLOWING STRUCTURAL FORMULA